EGFR (Epidermal Growth Factor Receptor) is normally embedded in the cell membrane on the cell surface. EGFR gene is 118 kb in length, includes 28 exons, and encodes a 170 kD glycoprotein comprised of 1186 amino acids. It is a membrane protein that plays an important role in regulating proliferation, growth, repair and survival of tumor cells. Currently, targeted therapy has become an important tool in the clinical treatment of Non Small Cell Lung Cancer (NSCLC). Iressa (Gefitinib, AstraZeneca) and Tarceva (Erlotinib, Roche), which function as EGFR tyrosine kinase inhibitors (TKI), are the main drugs approved by FDA for NSCLC targeted therapy. However, clinical experiments showed that Iressa and Tarceva only had significant therapeutic effects on 10-30% NSCLC patients. Further studies indicated that EGFR gene mutations relate to the therapeutic effects of NSCLC targeted therapy, and most of the patients carrying EGFR gene mutations showed significant therapeutic effects. A large number of research documents indicated that EGFR gene mutations are mainly located in the tyrosine kinase coding domain (exons 18-21), wherein deletion in exon 19 (746-753) accounts for about 45% of all mutations, and substitution in exon 21 (mainly L858R) accounts for about 40% of all mutations. At present, it is generally believed that these two hot mutations can enhance the sensitivity of tumor cells against TKI, and can be used as an effective index to predict TKI treatment. Therefore, the detection of EGFR gene mutations has an important reference value for guiding clinical administration in patients with NSCLC. Methods for detecting EGFR currently used in clinical treatment include: 1) traditional sequencing. This method has high accuracy. However, high requirement on the sample source, long sequencing time, the need for sequence analysis, and high cost of this method limit its use in clinic. 2) Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). It is a classical method for detecting gene mutations, and can detect unknown mutations. It has advantages of simple operation and high sensitivity. However, it also has an obvious disadvantage, which is the requirement for parallel standard control. Additionally, this method has a high false positive: the detection rate is 75-95% when the tested PCR product is less than 200 bp, while the detection rate is only about 50% when the tested PCR sample is more than 400 bp. 3) Mutation enriched PCR: a two-step PCR using a restriction endonuclease to selectively digest the wild type EGFR gene. After the first PCR, the wild type EGFR is digested selectively and the mutated EGFR genes are enriched, and then the second PCR is conducted. The PCR product is detected by electrophoresis, and whether EGFR is mutated is determined based on the detection results of PCR product. This method is highly sensitive, and can detect one mutation among 103-104 wild type EGFRs. However, this method needs twice PCR and enzyme digestion, thus is complex and time-consuming. In addition, there are technologies like AMRS and micro digital PCR, but their application in clinic still needs time.
Thus, there is an urgent need for a fast and efficient method for detecting EGFR gene mutations clinically. The inventor found, during the research of fragment DNA detection, a new method for detecting DNA fragments, including cyclizing and then amplifying DNA fragments. Based on this discovery and in combination with the second generation high throughput sequencing technology, the inventor improved the aforementioned method and designed optimized primers specifically based on the EGFR genes, and developed a method and a kit thereof for sequencing and analyzing EGFR gene mutations in plasma DNAs.